General limitations on commercial production of compounds include the efficiency of synthesis and, if a complex synthesis process is involved, the efficiency of recovery. Polypeptide production is an example of the limitations that can effect commercial production. The advent of recombinant DNA techniques has enabled investigators to produce substantial amounts of desired polypeptides in a variety of expression systems (e.g., bacterial, fungal, mammalian, yeast, plant and insect cells). Several problems typically occur, however, when such cell types are used as host cells for heterologous gene expression. For example, recombinant polypeptides produced by expression of heterologous genes in eukaryotic cells are often rapidly degraded by proteases in the cells. Those polypeptides that do manage to accumulate when expressed in a cell can be disruptive to the normal machinery of the cell, thereby lowering the growth and/or survival rate and the polypeptide production ability of the cell.
Recombinant polypeptides produced by expression of heterologous genes in bacterial cells are usually found in the insoluble or "inclusion body" fraction of a bacterial lysate, rendering the bacterial cells and the polypeptides useless for biological and biochemical applications. Such inclusion bodies typically require further manipulation in order to solubilize and re-fold the recombinant polypeptide under conditions that must be determined empirically, resulting in further uncertainties. Moreover, recombinant polypeptides produced in bacterial cells often have low activity due to a failure of the polypeptides to assume a natural conformation and/or the inability of cells to glycosylate such polypeptides.
To overcome these problems, prior investigators have concentrated on improving the expression of recombinant polypeptides in bacterial cells using various "fusion partners" linked to desired heterologous polypeptides to enable the expression and/or secretion of polypeptides in large amounts (U.S. Pat. No. 5,270,181, by McCoy et al., issued Dec. 14, 1993). Such fusion protein systems, however, fail to overcome the basic protein conformation problems encountered by expressing heterologous genes in a bacterial cell.
The use and purification of polypeptides produced by recombinant expression systems is a serious problem since recombinant production of polypeptides has become a mainstay in the development and production of therapeutic and diagnostic reagents. There is a continuing need for a simpler and commercially efficient method to produce substantial amounts of optimally active recombinant polypeptides for use in experimental, therapeutic and diagnostic applications.